Transferrin receptor specific antibody-neuropharmaceutical agent conjugates

ABSTRACT

The present invention pertains to a method for delivering a neuropharmaceutical or diagnostic agent across the blood brain barrier to the brain of a host. The method comprises administering to the host a therapeutically effective amount of an antibody-neuropharmaceutical or diagnostic agent conjugate wherein the antibody is reactive with a transferrin receptor. Other aspects of this invention include a delivery system comprising an antibody reactive with a transferrin receptor linked to a neuropharmaceutical or diagnostic agent and methods for treating hosts afflicted with a disease associated with a neurological disorder.

DESCRIPTION RELATED APPLICATIONS

This application is a Continuation-in-Part of U.S. application Ser. No.07/846,830 filed Mar. 6, 1992, now U.S. Pat. No. 5,182,107, which is aContinuation-in-Part of PCT/US90/05077, filed Sep. 7, 1990, designatingthe United States which, in turn, is a Continuation-in-Part of U.S.application Ser. No. 07/404,089 filed Sep. 7, 1989, now U.S. Pat. No.5,154,924.

BACKGROUND

The capillaries that supply blood to the tissues of the brain constitutethe blood brain barrier (Goldstein et al. (1986) Scientific American255:74-83; Pardridge, W. M. (1986) Endocrin. Rev. 7:314-330). Theendothelial cells which form the brain capillaries are different fromthose found in other tissues in the body. Brain capillary endothelialcells are joined together by tight intercellular junctions which form acontinuous wall against the passive movement of substances from theblood to the brain. These cells are also different in that they have fewpinocytic vesicles which in other tissues allow somewhat unselectivetransport across the capillary wall. Also lacking are continuous gaps orchannels running through the cells which would allow unrestrictedpassage.

The blood-brain barrier functions to ensure that the environment of thebrain is constantly controlled. The levels of various substances in theblood, such as hormones, amino acids and ions, undergo frequent smallfluctuations which can be brought about by activities such as eating andexercise (Goldstein et al, cited supra). If the brain were not protectedby the blood brain barrier from these variations in serum composition,the result could be uncontrolled neural activity.

The isolation of the brain from the bloodstream is not complete. If thiswere the case, the brain would be unable to function properly due to alack of nutrients and because of the need to exchange chemicals with therest of the body. The presence of specific transport systems within thecapillary endothelial cells assures that the brain receives, in acontrolled manner, all of the compounds required for normal growth andfunction. In many instances, these transport systems consist ofmembrane-associated receptors which, upon binding of their respectiveligand, are internalized by the cell (Pardridge, W. M., cited supra).Vesicles containing the receptor-ligand complex then migrate to theabluminal surface of the endothelial cell where the ligand is released.

The problem posed by the blood-brain barrier is that, in the process ofprotecting the brain, it excludes many potentially useful therapeuticagents. Presently, only substances which are sufficiently lipophilic canpenetrate the blood-brain barrier (Goldstein et al, cited supra;Pardridge, W. M., cited supra). Some drugs can be modified to make themmore lipophilic and thereby increase their ability to cross the bloodbrain barrier. However, each modification has to be tested individuallyon each drug and the modification can alter the activity of the drug.The modification can also have a very general effect in that it willincrease the ability of the compound to cross all cellular membranes,not only those of brain capillary endothelial cells.

SUMMARY OF THE INVENTION

The present invention pertains to a method for delivering aneuropharmaceutical or diagnostic agent across the blood brain barrierto the brain of a host. The method comprises administering to the host atherapeutically effective amount of an antibody-neuropharmaceutical ordiagnostic agent conjugate wherein the antibody is reactive with atransferrin receptor. The conjugate is administered under conditionswhereby binding of the antibody to a transferrin receptor on a braincapillary endothelial cell occurs and the neuropharmaceutical agent istransferred across the blood brain barrier in a pharmaceutically activeform. Other aspects of this invention include a delivery systemcomprising an antibody reactive with a transferrin receptor linked to aneuropharmaceutical agent and methods for treating hosts afflicted witha disease associated with a neurological disorder.

Presently available means for delivering therapeutic or diagnosticagents to the brain are limited in that they are invasive. The deliverysystem of the present invention is non-invasive and can utilize readilyavailable antibodies reactive with a transferrin receptor as carriersfor neuropharmaceutical agents. The delivery system is advantageous inthat the antibodies are capable of transporting neuropharmaceuticalagents across the blood brain barrier without being susceptible topremature release of the neuropharmaceutical agent prior to reaching thebrain-side of the blood brain barrier. Further, if the therapeuticactivity of the agent to be delivered to the brain is not altered by theaddition of a linker, a noncleavable linker can be used to link theneuropharmaceutical agent to the antibody.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graphic representation of rat brain uptake of ¹⁴ C-labelledmurine monoclonal antibody (OX-26) to rat transferrin receptor in ratswhere the percent injected dose of radiolabelled antibody per brain andper 55 μl of blood is plotted versus time post-injection.

FIG. 2 is a histogram illustrating time dependent changes in thedisposition of radiolabelled OX-26 between brain parenchyma andvasculature.

FIG. 3 is a histogram illustrating the enhanced delivery of methotrexateacross the blood-brain barrier when administered as a conjugate withOX-26.

FIGS. 4A-4C illustrate in three histograms (A,B and C) the distributionin the brain of both the antibody and the AZT components of an OX-26-AZTconjugate.

FIG. 5 is a histogram illustrating the experimental results of deliveryof a protein, horseradish peroxidase, across the blood-brain barrier inrat brains in the form of a conjugate with OX-26.

FIG. 6 is a histogram illustrating the experimental results ofdelivering soluble CD4 to rat brain parenchyma using CD4 in the form ofa conjugate with OX-26.

FIG. 7 is a histogram illustrating the biodistribution of antibody 128.1and control IgG in a cynomolgous monkey.

FIG. 8 is a histogram illustrating the uptake of NGF delivered as aconjugate with OX-26 into the cerebellum.

FIG. 9 is a histogram illustrating the effects of NGF delivered as aconjugate with OX-26 on the graft size of intraocular inplants.

DETAILED DESCRIPTION

The method for delivering a neuropharmaceutical agent across the bloodbrain barrier to the brain of a host comprises administering to the hosta therapeutically effective amount of an antibody-neuropharmaceuticalagent conjugate wherein the antibody is reactive with a transferrinreceptor present on a brain capillary endothelial cell. The method isconducted under conditions whereby the antibody binds to the transferrinreceptor on the brain capillary endothelial cell and theneuropharmaceutical agent is transferred across the blood brain barrierin a pharmaceutically active form.

The host can be an animal susceptible to a neurological disorder (i.e.,an animal having a brain). Examples of hosts include mammals such ashumans, domestic animals (e.g., dog, cat, cow or horse), mice and rats.

The neuropharmaceutical agent can be an agent having a therapeutic orprophylactic effect on a neurological disorder or any condition whichaffects biological functioning of the central nervous system. Examplesof neurological disorders include cancer (e.g. brain tumors), AutoimmuneDeficiency Syndrome (AIDS), stroke, epilepsy, Parkinson's disease,multiple sclerosis, neurodegenerative disease, trauma, depression,Alzheimer's disease, migraine, pain, or a seizure disorder. Classes ofneuropharmaceutical agents which can be used in this invention includeproteins, antibiotics, adrenergic agents, anticonvulsants, smallmolecules, nucleotide analogs, chemotherapeutic agents, anti-traumaagents, peptides and other classes of agents used to treat or prevent aneurological disorder. Examples of proteins include CD4 and superoxidedismutase (including soluble portions thereof), growth factors (e.g.nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF),ciliary neurotrophic factor (CNTF), neurotrophins 3,4 and 5 (NT-3, 4 and5) or fibroblast growth factor (FGF)), lymphokines or cytokines (e.g.interferon or interleukins (IL-2)) or antagonists thereof, neurotrophicfactors, dopamine decarboxylase and tricosanthin. A neurotrophic factoris defined as a factor capable of maintaining neuron survival or neuronregeneration or differentiation. The properties of the neurotrophicfactors are incorporated by reference herein from the followingreferences: (1) for ciliary neurotrophic factor (CNTF), see Manthorpe etal. (1986), Brain Research, 367: 282-286; Lin et al. (1989), Science246: 1023-1025; Stockli et al. (1989), Nature 342: 920-923; and Lam etal. (1991), Gene, 102: 271-276; (2) for brain-derived neurotrophicfactor (BDNF), see Barde et al (1987), Prog. Brain Res. 71: 185-189;Hofer et al. (1988), Nature 331: 261-262; and Leibrock et al (1989),Nature 334: 149-152; (3) for neurotrophin 3 (NT-3), see Hohn et al.(1990), Nature 344: 339-341; Maisonpierre et al (1990), Science 247:1446-1451; Rosenthal et al (1990), Neuron 4: 767-773; Ernfors et al.(1990), Proc. Natl. Acado Sci. USA 87: 5454-5458; Jones et al., (1990),Proc. Natl. Acad. Sci. USA 87: 8060-8064; and Kaisho et al (1990), FEBSLett. 266, 187-191; (4) for neurotrophin 4 (NT-4), see Hallbook et al(1991), Neuron 6: 845-858; and (5) for neurotrophin 5 (NT-5), seeBerkemeier et al (1991), Neuron 7: 857-866. Examples of antibioticsinclude amphotericin B, gentamycin sulfate, and pyrimethamine. Examplesof adrenergic agents (including blockers) include dopamine and atenolol.Examples of chemotherapeutic agents include adriamycin, methotrexate,cyclophosphamide, etoposide, and carboplatin. An example of ananticonvulsant which can be used is valproate and an anti-trauma agentwhich can be used is superoxide dismutase. Examples of peptides would besomatostatin analogues and enkephalinase inhibitors. Nucleotide analogswhich can be used include azidothymidine (hereinafter AZT),dideoxyinosine (ddI) and dideoxycytodine (ddC).

The antibody, which is reactive with a transferrin receptor present on abrain capillary endothelial cell, may also be conjugated to a diagnosticagent. In this method and delivery system, the neuropharmaceutical agentof the neuropharmaceutical agent--anti-transferrin receptor conjugatehas been replaced with a diagnostic agent. The diagnostic agent is thendelivered across the blood brain barrier to the brain of the host. Thediagnostic agent is then detected as indicative of the presence of aphysiological condition for which the diagnostic agent is intended. Forexample, the diagnostic agent may be an antibody to amyloid plaques.When conjugated to an antibody reactive with a transferrin receptorpresent on a brain capillary endothelial cell, this diagnostic agentantibody can be transferred across the blood brain barrier and can thensubsequently immunoreact with amyloid plaques. Such an immunoreaction isindicative of Alzheimer's Disease.

Serum transferrin is a monomeric glycoprotein with a molecular weight of80,000 daltons that binds iron in the circulation and transports it tothe various tissues(Aisen et al. (1980) Ann. Rev. Biochem. 49:357-393;MacGillivray et al. (1981) J. Biol. Chem. 258:3543-3553). The uptake ofiron by individual cells is mediated by the transferrin receptor, anintegral membrane glycoprotein consisting of two identical 95,000 daltonsubunits that are linked by a disulfide bond. The number of receptors onthe surface of a cell appears to correlate with cellular proliferation,with the highest number being on actively growing cells and the lowestbeing on resting and terminally differentiated cells. Jeffries et al(Nature Vol. 312 (November 1984) pp. 167-168) used monoclonal antibodiesto show that brain capillary endothelial cells have a high density oftransferrin receptors on their cell surface.

Antibodies which can be used within this invention are reactive with atransferrin receptor. The term antibody is intended to encompass bothpolyclonal and monoclonal antibodies. The preferred antibody is amonoclonal antibody reactive with a transferrin receptor. The termantibody is also intended to encompass mixtures of more than oneantibody reactive with a transferrin receptor (e.g., a cocktail ofdifferent types of monoclonal antibodies reactive with a transferrinreceptor). The term antibody is further intended to encompass wholeantibodies, biologically functional fragments thereof, and chimericantibodies comprising portions from more than one species, bifunctionalantibodies, etc. Biologically functional antibody fragments which can beused are those fragments sufficient for binding of the antibody fragmentto the transferrin receptor to occur.

The chimeric antibodies can comprise portions derived from two differentspecies (e.g., human constant region and murine variable or bindingregion). The portions derived from two different species can be joinedtogether chemically by conventional techniques or can be prepared assingle contiguous proteins using genetic engineering techniques. DNAencoding the proteins of both the light chain and heavy chain portionsof the chimeric antibody can be expressed as contiguous proteins.

The term transferrin receptor is intended to encompass the entirereceptor or portions thereof. Portions of the transferrin receptorinclude those portions sufficient for binding of the receptor to ananti-transferrin receptor antibody to occur.

Monoclonal antibodies reactive with at least a portion of thetransferrin receptor can be obtained (e.g., OX-26, B3/25 (Omary et al.(1980) Nature 286,888-891), T56/14 (Gatter et al. (1983) J. Clin. Path.36 539-545; Jefferies et al. Immunology (1985) 54:333-341), OKT-9(Sutherland et al. (1981) Proc. Natl. Acad. Sci. USA 78:4515-4519), L5.1(Rovera, C. (1982) Blood 59:671-678), 5E-9 (Haynes et al. (1981) J.Immunol. 127:347-351), RI7 217 (Trowbridge et al. Proc. Natl. Acad. Sci.USA 78:3039 (1981) and T58/30 (Omary et al. cited supra) or can beproduced using somatic cell hybridization techniques (Kohler andMilstein (1975) Nature 256, 495-497) or by other techniques. In atypical hybridization procedure, a crude or purified protein or peptidecomprising at least a portion of the transferrin receptor can be used asthe immunogen. An animal is vaccinated with the immunogen to obtain ananti-transferrin receptor antibody-producing spleen cells. The speciesof animal immunized will vary depending on the species of monoclonalantibody desired. The antibody producing cell is fused with animmortalizing cell (e.g. myeloma cell) to create a hybridoma capable ofsecreting anti-transferrin receptor antibodies. The unfused residualantibody-producing cells and immortalizing cells are eliminated.Hybridomas producing the anti-transferrin receptor antibodies areselected using conventional techniques and the selected anti-tranferrinreceptor antibody producing hybridomas are cloned and cultured.

Polyclonal antibodies can be prepared by immunizing an animal with acrude or purified protein or peptide comprising at least a portion of atransferrin receptor. The animal is maintained under conditions wherebyantibodies reactive with a transferrin receptor are produced. Blood iscollected from the animal upon reaching a desired titer of antibodies.The serum containing the polyclonal antibodies (antisera) is separatedfrom the other blood components. The polyclonal antibody-containingserum can optionally be further separated into fractions of particulartypes of antibodies (e.g. IgG, IgM).

The neuropharmaceutical agent can be linked to the antibody usingchemical conjugation techniques. Generally, the link is made via anamine or a sulfhydryl group. The link can be a cleavable link ornon-cleavable link depending upon whether the neuropharmaceutical agentis more effective when released in its native form or whether thepharmaceutical activity of the agent can be maintained while linked tothe antibody. The determination of whether to use a cleavable ornon-cleavable linker can be made without undue experimentation bymeasuring the activity of the drug in both native and linked forms orfor some drugs can be determined based on known activities of the drugin both the native and linked form.

For some cases involving the delivery of proteins or peptides to thebrain, release of the free protein or peptide may not be necessary ifthe biologically active portion of the protein or peptide is uneffectedby the link. As a result, antibody-protein or antibody peptideconjugates can be constructed using noncleavable linkers. Examples ofsuch proteins or peptides include CD4, superoxide dismutase, interferon,nerve growth factor, tricosanthin, dopamine decarboxylase, somatostatinanalogues and enkephalinase inhibitors. Terms such as "CD4" are usedherein to include modified versions of the natural molecule, such assoluble CD4, truncated CD4, etc. Examples of non-cleavable linkersystems which can be used in this invention include the carbodiimide(EDC), the sulfhydryl-maleimide, and the periodate systems. In thecarbodiimide system, a water soluble carbodiimide reacts with carboxylicacid groups on proteins and activates the carboxyl group. The carboxylgroup is coupled to an amino group of the second protein. The result ofthis reaction is a noncleavable amide bond between two proteins.

In the sulfhydryl-maleimide system, a sulfhydryl group is introducedonto an amine group of one of the proteins using a compound such asTraut's reagent. The other protein is reacted with an NHS ester (such asgamma-maleimidobutyric acid NHS ester (GMBS)) to form a maleimidederivative that is reactive with sulfhydryl groups. The two modifiedproteins are then reacted to form a covalent linkage that isnoncleavable.

Periodate coupling requires the presence of oligosaccharide groups oneither the carrier or the protein to be delivered. If these groups areavailable on the protein to be delivered (as in the case of horseradishperoxidase (HRP)), an active aldehyde is formed on the protein to bedelivered which can react with an amino group on the carrier. It is alsopossible to form active aldehyde groups from the carbohydrate groupspresent on antibody molecules. These groups can then be reacted withamino groups on the protein to be delivered generating a stableconjugate. Alternatively, the periodate oxidized antibody can be reactedwith a hydrazide derivative of a protein to be delivered which will alsoyield a stable conjugate.

The antibody-protein conjugate can also be produced as a contiguousprotein using genetic engineering techniques. Gene constructs can beprepared comprising DNA encoding the anti-transferrin receptor antibodyfused to DNA encoding the protein to be delivered across the blood brainbarrier. The protein can be expressed as a contiguous moleculecontaining both an antibody portion and a neuropharmaceutical agentportion.

Alternatively, the variable region of the antibody can be conjugated toproteins, such as factors, that can act as neuropharmaceutical agents.Since the variable region of antibodies immunologically interacts withthe complementary antigen, here the transferrin receptor, theseconjugates can be quickly delivered across the blood brain barrier. Thisrapid delivery occurs because the conjugates are less bulky than whenthe constant region of the antibody is present.

Cleavable linkers can be used to link neuro-pharmaceutical agents whichare to be deposited in the brain or when a non-cleavable linker altersthe activity of a neuropharmaceutical agent. Examples of cleavablelinkers include the acid labile linkers described in copending patentapplication Ser. No. 07/308,960 filed Feb. 6, 1989, the contents ofwhich are hereby incorporated by reference. Acid labile linkers includedisulfides such as N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP;Pharmacia), cis-aconitic acid, cis-carboxylic alkadienes, cis-carboxylicalkatrienes, and poly-maleic anhydrides. Other cleavable linkers arelinkers capable of attaching to primary alcohol groups. Examples ofneuropharmaceutical agents which can be linked via a cleavable linkinclude AZT, ddI, ddC, adriamycin, amphotericin B, pyrimethamine,valproate, methotrexate, cyclophosphamide, carboplatin and superoxidedimutase. The noncleavable linkers used generally to link proteins tothe antibody can also be used to link other neuropharmaceutical agentsto the antibody.

SPDP is a heterobifunctional crosslinking reagent that introducesthiol-reactive groups into either the monoclonal antibody or theneuropharmaceutical agent. The thiol-reactive group reacts with a freesulfhydryl group forming a disulfide bond.

In addition to covalent binding, conjugates can be formed employingnon-covalent bonds, such as those formed with bifunctional antibodies,ionic bonds, hydrogen bonds, hydropholic interactions, etc. Theimportant consideration is that the conjugate bond be strong enough toresult in passage of the conjugate through the blood-brain barrier.

The antibody-neuropharmaceutical agent conjugates can be administeredorally, by subcutaneous or other injection, intravenously,intramuscularly, parenternally, transdermally, nasally or rectally. Theform in which the conjugate is administered (e.g., capsule, tablet,solution, emulsion) will depend at least in part on the route by whichit is administered.

A therapeutically effective amount of an antibody-neuropharmaceuticalagent conjugate is that amount necessary to significantly reduce oreliminate symptoms associated with a particular neurological disorder.The therapeutically effective amount will be determined on an individualbasis and will be based, at least in part, on consideration of theindividuals's size, the severity of symptoms to be treated, the resultsought, the specific antibody, etc. Thus, the therapeutically effectiveamount can be determined by one of ordinary skill in the art employingsuch factors and using no more than routine experimentation.

Although the description above focuses on antibodies, any protein whichinteracts with the extracellular domain of the transferrin receptor,including the ligand binding site, could potentially serve as a vehiclefor the delivery of drugs across the blood-brain barrier. In addition toanti-transferrin receptor antibodies, this would include transferrin,the ligand which binds to the receptor, and any transferrin derivativeswhich retain receptor-binding activity. In fact, any ligand which bindsto the transferrin receptor could potentially be employed.

Conjugates between ligands and therapeutic or diagnostic agents can alsobe prepared where the ligands are reactive with other receptors, besidesthe transferrin receptor, which can also mediate the endocytotic ortranscytotic process of transporting macromolecules across theblood-brain barrier. These receptors are also on the cell surface of theendothelial cells which line brain capillaries. Among the receptor typesare those that react with insulin-like growth factors 1 or 2 (IGF 1 or2) or insulin itself. Other receptor types are those that react with lowdensity lipoprotein or with vasopressin. The ligands are thosesubstances which usually react with these receptors (e.g. IGF 1, IGF 2or insulin, low density liproprotein or vasopressin), derivatives ofthese substances which retain receptor-binding activity or antibodies tothese receptors. The properties of transferrin and other ligands whichbind to receptors on the cell surface of brain capillary endothelialcells are incorporated by reference herein from the followingreferences: (1) for transferrin, see Omary et al. (1981), J. Biol. Chem.256: 12888-12895; and McClelland et al. (1984), Cell 39: 267-274; (2)for insulin, see Pardridge et al. (1985), J. Neurochem. 44: 1771-1778;(3) for insulin-like growth factors, see Duffy et al. (1988), Metabolism37: 136-140; and Rosenfeld et al. (1987), Biochem. Biophys. Res. Comm.149: 159-166; (4) for low density lipoprotein, see Meresse et al.(1989), J. Neurochem. 53: 340-345; (5) and for vasopressin, see Zlokovicet al. (1990), Biochem. Biophys ACTA 1025: 191-198; Zlokovic et al.(1991), Am. J. Physiol. 260: F216-F224; and Zlokovic et al. (1991), Am.J. Physiol. 260: E633-E640. The therapeutic or diagnostic agents whichcan be conjugated to the ligands include the above-mentioned proteinssuch as nerve growth factor, superoxide dismutase, CD-4 or anti-amyloidantibody and drugs such as adriamycin, methotrexate or AZT.

The present invention will be illustrated by the following examples:

EXAMPLE 1

In Vitro Binding of Murine Monoclonal Antibodies to Human BrainEndothelial Cells

Two murine monoclonal antibodies, B3/25 and T58/30, described byTrowbridge (U.S. Pat. No. 4,434,156 issued Feb. 28, 1984, and NatureVol. 294, pp. 171-173 (1981)), the contents of both are herebyincorporated by reference, which recognize the human transferrinreceptor were tested for their ability to bind to human brain capillaryendothelial cells. Hybridoma cell lines which produce B3/25 and T58/30antibodies were obtained from the American Type Culture Collection(ATCC) in Rockville, Md., and grown in DMEM medium supplemented with 2.0mM glutamine, 10.0 mM HEPES (pH 7.2), 100 μM non-essential amino acidsand 10% heat-inactivated fetal calf serum. The hybridoma cultures werescaled-up in 225 cm² T-flasks for the production of milligram quantitiesof IgG antibody. The hybridoma supernatants were concentrated 50× usingvacuum dialysis and applied to a protein-A sepharose column using theBioRad MAPS buffer system. Purified antibody was eluted from the column,dialyzed against 0.1M sodium phosphate (pH 8.0), concentrated and storedin aliquots at -20° C.

Primary cultures of human brain endothelial cells were grown inflat-bottom 96-well plates until five days post-confluency. The cellswere then fixed using 3.0% buffered formalin and the plate blocked with1.0% bovine serum albumin (BSA) in Dulbecco's phosphate buffered saline(DPBS). Aliquots (100 μl) of the B3/25 or T58/30 antibodies, either inthe form of culture supernatants or purified protein, were then added tothe wells (antibody concentrations were in the range of 1-50 μg/ml).Antibody which had specifically bound to the fixed cells was detectedusing a biotin-labeled polyclonal goat-anti-mouse IgG antisera followedby a biotinylated horseradish peroxidase (HRP)/avidin mixture (AvidinBiotin Complex technique). Positive wells were determined using aTitertek Multiscan Enzyme Linked Immunosorbent Assay (ELISA) platereader. The results showed that both antibodies bind to human braincapillary endothelial cells with the T58/30 antibody exhibiting a higherlevel of binding.

These same antibodies were also tested for binding to human braincapillaries using sections of human brain tissue that were fresh frozen(without fixation), sectioned on a cryostat (section thickness was 5-20μm), placed on glass slides and fixed in acetone (10 minutes at roomtemperature). These sections were then stored at -20° C. prior to use.

The slides containing the human brain sections were allowed to come toroom temperature prior to use. The sections were then rehydrated in DPBSand incubated in methanol containing 0.3% H₂ O₂ to block endogenousperoxidate activity. The sections were blocked for fifteen minutes in asolution containing 0.2% non-fat dry milk and 0.2%methylmannopyranoside. B3/25 and T58/30 antibodies, purified asdiscussed above, were applied to the sections at a concentration of 5-50μg/ml and incubated at room temperature for one to two hours. Antibodythat specifically bound to the tissue was detected using theAvidin-Biotin Complex (ABC) technique as described above for the ELISAassay. Staining of capillaries in the human brain sections was observedwith both the B3/25 and T58/30 antibodies. The T58/30 antibody alsodisplayed some binding to the white matter of the brain cortex.

EXAMPLE 2

In-Vitro Binding of Murine Monoclonal Antibody OX-26 to Rat TransferrinReceptor

The OX-26 murine antibody, which recognizes the rat transferrinreceptor, has been shown in vivo to bind to brain capillary endothelialcells (Jeffries et al., cited supra). The murine hybridoma line whichproduces the OX-26 murine antibody was obtained and the hybridoma cellline was grown in RPMI 1640 medium supplemented with 2.0 mM glutamineand 10% heat-inactivated fetal calf serum. The OX-26 antibody waspurified using the affinity chromatography technique described inExample 1.

The purified antibody was tested in vitro as described for theanti-human transferrin receptor antibodies in Example 1 to determinewhether it would bind to brain capillaries in fresh frozen,acetone-fixed rat brain sections. The results showed that the OX-26anti-transferrin receptor antibody did bind to capillaries in rat brainsections in vitro.

EXAMPLE 3

In-Vivo Binding of OX-26 Murine Monoclonal Antibody to Rat TransferrinReceptor

Dose Range

The anti-rat transferrin receptor antibody OX-26 was tested in vivo byinjecting purified antibody (purification as described in Example 1)into female Sprague-Dawley rats (100-150 gm) via the tail vein. Prior toinjection, the rats were anesthetized with halothane. The samples,ranging from 2.0 mg to 0.05 mg of antibody/rat were injected into thetail vein in 400 μl aliquots. All doses were tested in duplicateanimals. One hour post-injection, the animals were sacrificed andperfused through the heart with DPBS to clear the blood from the organs.Immediately after the perfusion was completed, the brain was removed andquick frozen in liquid nitrogen. The frozen brain was then sectioned(30-50 μm) on a cryostat and the sections placed on glass microscopeslides. The brain sections were air dried at room temperature one to twohours before fixation in acetone (10 minutes at room temperature). Afterthis treatment the sections could be stored at -20° C.

The OX-26 antibody was localized in the brain sections usingimmunohistochemistry as described above for the in vitro experiments inExample 1. The addition of the primary antibody was unnecessary in thatit is present in the brain sections. The results indicated that theOX-26 antibody binds to rat brain capillary endothelial cells and thatdoses of as little as 50 μg result in detectable levels of antibody inthe brain using the methods described herein. Doses above 0.5 mg did notappear to show significantly more antibody binding to the endothelialcells, suggesting that the sites for antibody binding may be saturated.No specific binding to capillary endothelium was detected in the liver,kidney, heart, spleen or lung.

A non-specific antibody of the same subclass as OX-26 (IgG 2a) was alsotested in vivo to show that the binding of OX-26 to rat brainendothelial cells that has been observed is specific to the OX-26antibody. 0.5 mg of the control antibody was injected per rat asdescribed above. The results indicate that the staining pattern observedwith the OX-26 antibody is specific to that antibody.

Time Course

After establishing that the OX-26 antibody is detectable in the ratbrain capillaries after in vivo administration, the time frame in whichthis binding occurred was determined. Using 0.5 mg of purified OX-26antibody as the standard dose, brain sections taken from animalssacrificed 5 minutes, 15 minutes, 1 hour, 2 hours, 4 hours, 8 hours and24 hours post-injection were examined for the presence of OX-26antibody. All doses were administered in 400 μl aliquots and each timepoint was tested in duplicate animals. Samples were injected and therats were processed at the various times post-injection as describedabove in the dose range section.

The results showed that the OX-26 antibody can be detected in or on therat brain capillary endothelial cells as early as five minutes and aslate as 24 hours post-injection. At 4 and 8 hours post-injection, thestaining pattern of the antibody is very punctate suggesting that theantibody has accumulated in vesicular compartments either in endothelialor perivascular cells.

EXAMPLE 4

The Use of a Conjugate of OX-26 Murine Monoclonal Antibody forTranferring Horseradish Peroxidase Across the Blood Brain Barrier

Horseradish Peroxidase (HRP; 40 kD) was chosen as a compound to bedelivered to the brain because it is similar in size to severaltherapeutic agents and it can be easily detected in the brain using anenzymatic assay. HRP was conjugated to the OX-26 antibody using anon-cleavable periodate linkage and the ability of the antibody tofunction as a carrier of compounds to the brain was examined. Theantibody conjugate was tested in vivo to determine if the antibody coulddeliver HRP to the brain.

The antibody (10 mg) was first dialyzed overnight against 0.01M sodiumbicarbonate (pH 9.0). The HRP (10 mg) was dissolved in 2.5 ml deionizedwater, 0.1M sodium periodate (160 μl) was added and the mixture wasincubated for five minutes at room temperature. Ethylene glycol (250 μl)was added to the HRP solution followed by an additional five minuteincubation. This solution was then dialyzed overnight against 1.0 mMsodium acetate buffer (pH 4.4). To the dialyzed OX-26 antibody (2.0 ml,5.08 mg/ml) was added 200 μl of 1.0M sodium bicarbonate buffer, pH 9.5and 1.25 ml of the dialyzed HRP solution. This mixture was incubated inthe dark for two hours followed by the addition of 100 μl of 10 mg/mlsodium borohydride. The resulting mixture was incubated two additionalhours in the dark at 4° C. The protein was precipitated from thesolution by the addition of an equal volume of saturated ammoniumsulfate and resuspended in a minimal volume of water. Free antibody wasremoved from the mixture by chromatography on a concanavalin A-sepharosecolumn (a column which binds HRP and the HRP-antibody conjugate andallows the free antibody to pass through). The free HRP was removed bychromatography on a protein A-sepharose column which retains theantibody-HRP conjugate. The final product had an HRP/antibody ratio of4/1.

A time course experiment identical to that described in Example 3 wasperformed using the antibody-HRP conjugate. The antibody-HRP conjugate(0.5 mg) was injected in a 400 μl aliquot/rat. The animals weresacrificed at the various times post-injection and the brains processedas described above in Example 3. The antibody HRP conjugate waslocalized in the brain either by staining for antibodyimmunohistochemically as described in Example 1 or by directly stainingthe brain sections for the presence of HRP. To detect HRP, the slideswere first allowed to come to room temperature before incubating inmethanol for thirty minutes. The brain sections were then washed in DPBSand reacted with 3,3'-diamino benzidine (DAB), the substrate for HRP.The results showed that the OX-26 antibody HRP conjugate binds to ratbrain capillary endothelial cells in a manner identical to that of theunconjugated antibody. The punctate staining 4-8 hours after injectionwhich was seen with the antibody alone is also seen with the antibodyconjugate, suggesting that the conjugate can also be going into thepericytes on the abluminal side of the blood brain barrier. Takentogether, these results indicate that the OX-26 antibody can deliver aprotein molecule of at least 40 KD to the brain.

EXAMPLE 5

The In-Vivo Delivery of Adriamycin to the Brain by Murine MonoclonalAntibody OX-26

A non-cleavable linker system similar to that used in Example 4, wasused to couple the chemotherapeutic drug adriamycin to the OX-26antibody. The availability of antibodies that can detect adriamycin,aswell as the system previously described in Example 1 for detecting theantibody carrier allowed the use of immunohistochemical techniques formonitoring the localization of the antibody carrier as well as thedelivery of adriamycin to the brain.

To conjugate adriamycin to the antibody, the drug (10 mg in 0.5 ml DPBS)was oxidized by the addition of 200 μl of 0.1M sodium periodate. Thismixture was incubated for one hour at room temperature in the dark. Thereaction was quenched by the addition of 200 μl of ethylene glycolfollowed by a five minute incubation. The OX-26 antibody (5.0 mg in 0.5ml of carbonate buffer (pH 9.5)) was added to the oxidized adriamycinand incubated at room temperature for one hour. Sodium borohydride (100μl of 10 mg/ml) was added and the mixture was incubated for anadditional two hours at room temperature. The free adriamycin wasseparated from the OX-26 antibody-adriamycin conjugate by chromatographyon a PD-10 column. The adriamycin/OX-26 antibody ratio within theconjugate was 2/1. for this particular batch of conjugate.

The effectiveness of the OX-26 antibody as a carrier for deliveringadriamycin to the brain was determined by administering 0.5 mg of theantibody-adriamycin conjugate in a 400 μl aliquot per rat by injectionvia the tail vein. One hour post-injection, the rat was sacrificed andthe brain processed as described in Example 1. All injections wereperformed in duplicate. As a control, 400 μg of free adriamycin in a 400μl aliquot was also injected into a rat. Immunohistochemistry was usedto detect both the carrier OX-26 antibody and the adriamycin in the ratbrain sections. In the case of adriamycin, polyclonal rabbitanti-adriamycin antisera was applied to the sections followed by abiotinylated goat anti-rabbit IgG antisera. This was then followed bythe addition of a biotinylated HRP/avidin mixture and enzymaticdetection of HRP.

The results indicate that both the OX-26 antibody and the conjugatedadriamycin localized to the rat brain capillary endothelial cells afterin vivo administration. There is no evidence that free adriamycin bindsto brain capillary endothelial cells or enters the brain.

An adriamycin-OX-26 conjugate coupled via a carbodiimide linkage wasalso synthesized (drug/ antibody ratio of 10/1) and tested in vivo. Theresults of this experiment were essentially identical to that obtainedwith the periodate-linked antibody-drug conjugate. In both cases,staining for the antibody carrier was quite strong and was visualized inthe capillaries in all areas of the brain. This staining was evenlydistributed along the capillaries. Staining for adriamycin was lessintense but again was seen in capillaries throughout the brain. Somepunctate staining was observed which suggests accumulation in pericyteswhich lie on the brain side of the blood-brain barrier.

EXAMPLE 6

In Vivo Delivery of Methotrexate to the Brain by Murine MonoclonalAntibody OX-26.

A noncleavable carbodiimide linkage was used to couple methotrexate tothe OX-26 murine monoclonal antibody. A system analogous to thatdescribed in Example 5 was used to monitor the delivery of both themethotrexate and the carrier antibody to the brain capillary endothelialcells.

Methotrexate was coupled to murine monoclonal antibody OX-26 via itsactive ester. Briefly, 81 mg (0.178 mM) of methotrexate (Aldrich) wasstirred with 21 mg (0.182 mM) of N-hydroxysuccinimide (Aldrich) in 3 mlof dimethylformamide (DMF) at 4° C.Ethyl-3-dimethylaminopropyl-carbodiimide (180 mg;EDC;0.52mM) was addedto this solution and the reaction mixture was stirred overnight. Thecrude ester was purified from the reaction by-products by flashchromatography over silica gel 60 (Merck) using a solution of 10%methanol in chloroform as an eluant. The purified active ester fractionswere pooled and concentrated to dryness. The ester was dissolved in 1 mlof DMF and stored at -20° C. until use. 50 mg (50%) of active ester wasrecovered as determined by A₃₇₂ (ε_(b) 372 =7200).

A solution of OX-26 containing 2.1 mg (14 nmoles) of antibody in 0.9 mlof 0.1M phosphate (pH 8.0) was thawed to 4° C. To this stirred antibodysolution was added 1.4 μL (140 nmoles) of the active ester prepared asdescribed above. After 16 hours at 4° C., the mixture waschromatographed over Sephadex PD-10 column (Pharmacia) using phosphatebuffered saline (PBS) to separate conjugate from free drug. Thefractions containing the antibody-methotrexate conjugate were pooled.Antibody and drug concentration were determined spectrophotometricallyas described by Endo et al. (Cancer Research (1988) 48:3330-3335). Thefinal conjugate contained 7 methotrexates/antibody.

The ability of the OX-26 monoclonal antibody to deliver methotrexate tothe rat brain capillary endothelial cells was tested in vivo byinjecting 0.2 mg of conjugate (in 400 μl) into each of two rats via thetail vein. The animals were sacrificed one hour post-injection and thebrains processed for immunohistochemistry as described in Example 1. Todetect methotrexate in the brain, a rabbit antisera raised againstmethotrexate was used as the primary antibody. A biotinylatedgoat-anti-rabbit antisera in conjunction with a mixture of biotinylatedHRP and avidin was then used to visualize methotrexate in the rat brain.The carrier antibody was detected as described previously.

The results of these experiments indicate that methotrexate in the formof a conjugate with OX-26 does accumulate along or in the capillaryendothelial cells of the brain. The staining observed for methotrexateis comparable in intensity to that seen for the carrier. The stainingappears to be in all areas of the brain and is evenly distributed alongthe capillaries.

EXAMPLE 7

Antibody Derivatives

The Fc portion of the OX-26 murine monoclonal antibody was removed todetermine whether this would alter its localization to or uptake by therat brain capillary endothelial cells. Fab fragments were produced fromintact IgG's via digestion with papain. F(ab)₂ fragments of OX-26 wereproduced from intact IgG's via digestion with pepsin. Kits availablefrom Pierce Chemical Co. contain the reagents and protocols for cleavingthe antibody to obtain the fragments. The F(ab')₂ fragment (0.2 mgdoses) in 400 μl aliquots were injected into rats via the tail vein. Atime course experiment identical to that done with the intact antibody(Example 2) was then performed. F(ab')₂ fragment was detectedimmunohistochemically using a goat anti-mouse F(ab')₂ antisera followedby a biotinylated rabbit anti-goat IgG antisera. A biotinylatedHRP/avidin mixture was added and the antibody complex was visualizedusing an HRP enzymatic assay. The results indicate that the F(ab)₂fragment of the OX-26 antibody binds to the capillary endothelial cellsof the rat brain.

EXAMPLE 8

Measurement of OX-26 in Brain Tissue

To quantitate the amount of OX-26 which accumulates in the brain,radioactively-labelled antibody was injected into rats via the tailvein. Antibodies were labelled with either ¹⁴ C-acetic anhydride or ³H-succinimidyl propionate essentially as described in Kummer, U.,Methods in Enzymology, 121: 670-678 (1986), Mondelaro, R. C., andRueckert, R. R., J. of Biological Chemistry, 250: 1413-1421 (1975),hereby incorporated by reference. For all experiments, the radiolabelledcompounds were injected as a 400 μl bolus into the tail vein of femaleSprague-Dawley rats (100-125 gms) under Halothane anesthesia and theanimals were sacrificed at the appropriate time post-injection using alethal dose of anesthetic. A ³ H-labelled IgG2a control antibody wasco-injected with the ¹⁴ C-labelled OX-26 to serve as a control fornon-specific radioactivity in the brain due to residual blood. At theappropriate time post-injection, animals were sacrificed and the brainswere removed immediately and homogenized in 5 ml of 0.5% sodiumdodecylsulfate using an Omni-mixer. An aliquot of the homogenate wasincubated overnight with 2 ml of Soluene 350 tissue solubilizer prior toliquid scintillation counting. All data were collected asdisintegrations per minute (dpm). Blood samples were centrifuged topellet red blood cells (which do not display significant binding ofradiolabelled materials) and the radioactivity in an aliquot of serumdetermined using liquid scintillation counting.

The amount of antibody associated with the brain was determined atvarious times post-injection to examine the pharmacokinetics of brainuptake. In addition, the amount of labelled antibody in the blood wasmeasured so that the rate of clearance from the bloodstream could bedetermined. This information was also used to calculate the amount ofradioactivity in the brain due to blood contamination, which was thensubtracted from the total to give the amount of antibody that isspecifically associated with the brain.

A peak level of ¹⁴ C-labelled OX-26 corresponding to approximately 0.9%of the injected dose was reached in the brain between 1 and 4 hourspost-injection as illustrated in FIG. 1 (with the values shown as meansplus or minus standard error of the mean (SEM) and N=3 rats per timepoint). The amount of radioactivity associated with the brain decreasedsteadily from 4 to 48 hours post-injection, at which point it leveledoff at approximately 0.3% of the injected dose. The accumulation ofOX-26 in the brain was significantly reduced by the addition ofunlabelled monoclonal antibody (0.5 or 2.0 mg in the bolus injection).As an additional control, a ³ H-IgG2a control antibody was co-injectedwith the ¹⁴ C-OX-26. The control antibody did not accumulate in thebrain and represented the blood contamination of the brain.

In contrast to the levels in the brain, the blood level of OX-26 droppedquite dramatically immediately after injection such that by 1 hourpost-injection, the percent of injected dose in 55 μl of blood (thevolume of blood associated with the brain) was approximately 0.16% asillustrated in FIG. 1. This corresponds to a value of approximately 20%of the injected dose in the total blood volume of the rat. Extraction oftotal IgG from serum followed by polyacrylamide gel electrophoresis(PAGE) and autoradiography did not reveal detectable levels of OX-26degradation indicating that the antibody remains intact in the blood aslong as 48 hours after injection.

EXAMPLE 9

Distribution of OX-26 in Brain Parenchyma and Capillaries

To demonstrate that anti-transferrin receptor antibody accumulates inthe brain parenchyma, homogenates of brains taken from animals injectedwith labelled OX-26 were depleted of capillaries by centrifugationthrough dextran to yield a brain tissue supernatant and a capillarypellet. Capillary depletion experiments followed the procedure ofTriguero, et al., J. of Neurochemistry, 54: 1882-1888 (1990), herebyincorporated by reference. As for the brain uptake experiments ofExample 8, the radiolabelled compounds were injected as a 400 μl bolusinto the tail vein of females Sprague-Dawley rats (100-125 gm) underHalothane anesthesia and the animals were sacrificed at the appropriatetime post-injection using a lethal dose of anesthetic. A ³ H-labelledIgG 2a control antibody was co-injected with the ¹⁴ C-labelled OX-26 toserve as a control for non-specific radioactivity in the brain due toresidual blood. After sacrifice, the brains were removed and kept onice. After an initial mincing, the brains were homogenized by hand (8-10strokes) in 3.5 ml of ice cold physiologic buffer (100 mM NaCl, 4.7 mMKCl, 2.5 mM CaCl₂, 1.2 mM KH₂ PO₄, 1.2 mM MgSO₄, 14.5 mM HEPES, 10 mMD-glucose, pH 7.4). Four ml of 26% dextran solution in buffer was addedand homogenization was continued (3 strokes). After removing an aliquotof the homogenate, the remainder was spun at 7200 rpm in a swingingbucket rotor. The resulting supernatant was carefully removed from thecapillary pellet. The entire capillary pellet and aliquots of of thehomogenate and supernatant were incubated overnight with 2 ml of Soluene350 prior to liquid scintillation counting. This method removes greaterthan 90% of the vasculature from the brain homogenate (Triguero et al.,cited supra).

A comparison of the relative amounts of radioactivity in the differentbrain fractions as a function of time indicates whether transcytosis ofthe labelled antibody has occurred. The amount of OX-26 in total brainhomogenate, the brain parenchyma fraction and the brain capillaryfraction at an early time (30 minutes) and a later time (24 hours)post-injection is illustrated in FIG. 2. The values in FIG. 2 are shownas means±SEM with N=3 rats per time point. At the 30 minute time point,more of the radiolabelled antibody is associated with the capillaryfraction than with the brain parenchyma fraction (0.36% of the injecteddose (%ID) and 0.23% ID, respectively). By 24 hours post-injection, thedistribution is reversed and the majority of the radioactivity (0.36%ID) is in the parenchymal fraction as compared to the capillary fraction(0.12% ID). The redistribution of the radiolabelled OX-26 from thecapillary fraction to the parenchyma fraction is consistent with thetime dependent migration of the anti-transferrin receptor antibodyacross the blood-brain barrier.

EXAMPLE 10

Distribution of an OX-26-methotrexate Conjugate in Brain Parenchyma andCapillaries

Capillary depletion studies following the procedures described inExample 9 were performed with an OX-26-methotrexate (MTX) conjugatelinked via a gamma-hydrazid as described in Kralovec, et al., J. ofMedicinal Chem., 32: 2426-2431 (1989), hereby incorporated by reference,in which the MTX moiety was labelled with ³ H. As with unconjugatedantibody, the amount of label in the capillary fraction at 30 minutespost-injection is greater than the parenchyma fraction (approximately2-fold as illustrated in FIG. 3, with the data expressed as means±SEMand N=3 rats per time point). This distribution changes over time suchthat by 24 hours post-injection, approximately 4.5-fold more of thelabelled MTX is in the brain parenchyma than in the capillaries. Theseresults are consistent to those obtained with unconjugated antibody and,again, suggest that these compounds cross the blood-brain barrier.

To ensure that these results were not due to contaminating amounts offree ³ H-MTX or ³ H-MTX that had been cleaved from the conjugate afterinjection, a co-mix of labelled drug and antibody was injected into ratsand a capillary depletion experiment performed. The amount of ³ H-MTX inthe different brain fraction is significantly lower for the co-mix ascompared to the conjugate (as much as 47 fold in the case of thecapillary fraction at 30 minutes post-injection as illustrated in FIG.3). The ³ H-MTX and the co-mix also does not show the change indistribution of the label between the different brain fractions overtime as was seen with the antibody-MTX conjugate or antibody alone.These results demonstrate that delivery of ³ H-MTX across theblood-brain barrier to the brain parenchyma is greatly enhanced by theconjugation of the drug to the anti-transferrin receptor antibody OX-26.

EXAMPLE 11

Distribution of OX-26-AZT in Brain Parenchyma and Capillaries

Capillary depletion studies following the procedures of Example 9 wereperformed with an OX-26-AZT conjugate using a pH-sensitive succinatelinker. These studies employed a dual-labelled conjugate in which theAZT was ¹⁴ C-labelled and the antibody carrier was ³ H-labelled. The useof such a conjugate allowed independent monitoring of the disposition ofboth the antibody and AZT within the brain.

The linker was synthesized as follows. Succinic anhydride was used toacylate the AZT by reacting equimolar amounts of these two compounds for3 hours at room temperature under argon in the presence ofdimethylaminopyridine and sodium bisulfate in freshly distilledpyridine. The product was isolated by chromatography on a DEAE sephadexA50 column run with a triethylammonium bicarbonate buffer. The succinatederivative of AZT was activated at the carboxyl group as the NHS esterby reaction with equimolar amounts of N-hydroxysuccinimide anddicyclohexylcarbodiimide (DCC) in freshly distilled THF at 4° C. for 2hours. The product was purified by flash charomatography on silica gel.The resulting NHS-ester of AZT-succinate was used to acylate aminegroups on OX-26, resulting in an AZT-OX-26 conjugate. A 15-fold molarexcess of AZT-NHS ester was reacted with OX-26 in HEPES buffer overnightat 4° C. The antibody-drug conjugate was isolated from free drug on aPD-10 column. The molar ratio of drug to antibody was 7:1. These studiesemployed a dual-labelled conjugate in which the AZT was ¹⁴ C-labelledand the antibody carrier was ³ H-labelled.

Similar levels of OX-26 and AZT are seen in the capillary fraction ofthe brain and these levels decrease with time, suggesting that thematerials are not being retained by the capillary endothelial cells asillustrated in FIG. 4c. As the levels of OX-26 in the capillary fractiondecrease, the levels in the parenchyma fraction increase, indicatingthat the antibody is migrating from the capillaries to the parenchyma ina time-dependent manner as illustrated in FIG. 4b. In contrast, thelevels of AZT in the brain parenchyma do not rise significantly,suggesting that the majority of the drug is released in the endothelialcells and is not transported across the blood-brain barrier. The levelsof OX-26 and AZT remained similar in unfractionated homogenates overtime as illustrated in FIG. 4a. The data in FIG. 4 are expressed asmeans±SEM with N=3 rats per time point. These results indicate that thelinker is cleaved within the endothelial cells and may represent amethod for delivering compounds to those cells.

EXAMPLE 12

Distribution of OX-26-Horseradish. Peroxidase (HRP) in Brain Parenchymaand Capillaries

Capillary depletion studies following the procedures described for OX-26in Example 9 were performed with a ³ H-labelled OX-26-HRP conjugate thatwas prepared using a non-cleavable periodate linkage as described inExample 4. The tritium label was distributed between the antibody andthe HRP portion of the conjugate. At 1 hour post-injection, the majorityof the radioactivity associated with the brain is in the capillaryfraction as illustrated in FIG. 5. The data in FIG. 5 are expressed asmeans±SEM with N=3 rats per time point. By 4 hours post-injection, thedistribution of radioactivity associated with the brain changed suchthat the majority is in the fraction which represents the brainparenchyma. At 24 hours post-injection, essentially all of the ³H-labelled OX-26-HRP conjugate is in the parenchyma fraction of thebrain indicating that the material has crossed the blood-brain barrier.Similar results were obtained in experiments in which only the HRPportion of the conjugate was radiolabelled.

The percent of injected dose of the OX-26-HRP conjugate that reaches thebrain is somewhat lower than that for antibody alone. This is mostlikely due to the presence of 2 to 3 40 kD HRP molecules attached toeach carrier and that these "passenger" molecules are randomly attachedto the carrier. Due to this, many of the HRP passengers may be attachedto the antibody in such a way as to interfere with antigen recognition.This problem can be alleviated by directing the attachment of thepassenger to regions of the carrier removed from critical functionaldomains.

EXAMPLE 13

Distribution of OX-26-CD4 in Brain Parenchyma and Capillaries

A soluble form of CD4, consisting of amino acids 1-368, was conjugatedto OX-26 using a linkage that directed the attachment of the CD4 to thecarbohydrate groups located in the Fc portion of the antibody. Bydirecting the site of attachment in this way, the chance that thepassenger molecules will interfere with antibody-antigen recongition islessened. The linkage between the proteins was achieved by firstintroducing a sulfhydryl group onto CD4 using SATA (N-SuccinimidylS-acetylthioacetate), a commerically available compound. A hydrizidderivative of SPDP, another commercial cross-linking agent, was attachedto OX-26 via carbohydrate groups on the antibody. Reaction of the twomodified proteins gives rise to a disulfide-linked conjugate.

More specifically the linkage between the proteins was achieved by firstintroducing a sulfhydryl group onto CD4 using N-succinimidylS-acetylthioacetate (SATA), a commercially available compound. A 4-foldmolar excess of SATA was added to 5 mg of CD4 in 0.1M sodium phosphatebuffer containing 3 mM EDTA (pH 7.5). This mixture was reacted at roomtemperature in the dark for 30 minutes. Unreacted starting materialswere removed by passage over a PD-10 column. A hydrizid derivative ofSPDP, another commercially available cross-linking agent, was attachedto OX-26 via carbohydrate groups on the antibody. Ten milligrams ofOX-26 in 2.0 ml of 0.1M sodium acetate, 0.15M sodium chloride (pH 5.0)was reacted with a 1000-fold molar excess of sodium periodate for 1 hourat 4° C. in the dark. Unreacted starting materials were removed bypassage over a PD-10 column. The oxidized antibody was reacted with a30-fold molar excess of hydrazido-SPDP overnight at 4° C. with stirring.Reaction of the two modified proteins gives rise to a disulfide-linkedconjugate. One tenth volume of 0.5M hydroxylamine was added to thethioacetylated CD4 (CD4-DATA) and derivatized antibody was then addedsuch that the ratio of CD4 to antibody was 7.5:1. This mixture wasreacted at room temperature in the dark for 2 hours. Conjugate waspurified by running the reaction mixture over a protein A columnfollowed by a CD4 affinity column.

Capillary depletion experiments following the procedures described inExample 9 with OX-26 were performed with an OX-26-CD4 conjugate in whichonly the CD4 portion was ³ H-labelled. Time dependent changes in thedistribution of the labelled conjugate between the capillary andparenchyma fractions of the brain which are consistent with transcytosisacross the blood-brain barrier were observed as illustrated in FIG. 6.The data in FIG. 6 are expressed as means±SEM with N=3 rats per timepoint.

EXAMPLE 14

Biodistribution and Brain Uptake of Anti-Human Transferrin ReceptorAntibodies in Cynomolgous Monkeys

A collection of 32 murine monoclonal antibodies which recognize variousepitopes on the human transferrin receptor were examined for reactivitywith brain capillary endothelial cells in sections from human, monkey(cynomolgous), rat and rabbit brain samples by the immunohistochemicalmethods described in Example 1. These antibodies were obtained from Dr.Ian Trowbridge of the Salk Institute, LaJolla, Calif. All 32 antibodiesdisplayed some reactivity with human brain endothelial cells. Twoantibodies reacted very weakly with rabbit brain capillaries and nonereacted with rat. While 21 of the antibodies reacted with monkey braincapillaries, only 2 displayed strong reactivity comparable to that seenwith human brain capillaries. These 2 antibodies are herewithin referredto as 128.1 and Z35.2.

These antibodies were used to determine the tissue distribution andblood clearance of the ¹⁴ C-labelled anti-human transferrin receptorantibodies 128.1 and Z35.2 in 2 male cynomolgous monkeys. 128.1 or Z35.2was administered concurrently with a ³ H-labelled control IgG to one ofthe monkeys with an intravenous catheter. During the course of thestudy, blood samples were collected to determine the clearance of theantibodies from the circulation. At 24 hours post-injection, the animalswere euthanized and selected organs and representative tissues werecollected for the determination of isotope distribution and clearance bycombustion. In addition, samples from different regions of the brainwere processed as described for the capillary depletion experiments inExample 9 to determine whether the antibodies had crossed theblood-brain barrier. The results of the capillary depletion experimentswere performed on samples from the cortex, frontal cortex, cerebellumand striatum. All samples had greater than 90% of the 128.1 or Z35.2 inthe brain parenchyma, suggesting that the antibodies crossed theblood-brain barrier. The levels of the control antibody in the samesamples were from 5 to 10-fold lower. Using the average brain homogenatevalue for dpm/G tissue, the percent injected dose of 128.1 in the wholebrain is approximately 0.2-0.3%. This compares to a value of 0.3-0.5%for OX-26 in the rat at 24 hours post-injection. A comparison of theratios of 128.1 to the control antibody for various organs isillustrated in FIG. 7. Similar results were obtained for Z35.2. Theseresults suggest that 128.1 is preferentially taken up by the brain ascompared to control antibody. For the majority of organs and tissuestested, the ratio of 128.1 to control is less than 2.

EXAMPLE 15

Delivery of Nerve Growth Factor to the Brain via Anti-TransferrinReceptor Antibodies and Antibody Fragments

Nerve growth factor (NGF) is a 26,000 dalton protein which has beenshown in vitro and in vivo to support the growth of basal forebraincholinergic neurons. In Alzheimer's Disease, these cells undergosignificant degenerative changes which, at least in part, may beresponsible for some of the cognitive and memory deficits that areassociated with this disorder. Because of this correlation, NGF has beenproposed as a potential therapeutic agent for the treatment ofAlzheimer's Disease. The studies described below demonstrate the abilityof OX-26 to deliver NGF across the blood-brain barrier.

CONJUGATE SYNTHESIS AND PURIFICATION

The following procedure was followed for producing conjugates of NGF tothe OX-26 antibody or its Fab or F(ab')₂ fragments.

NGF was conjugated to the OX-26 antibody through a disulfide bond. NGFwas modified through its carboxyl groups with EDC(1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide) and PDP(pyridyldithiopropionate)-hydrazide to introduce a thiol-reactivepyridyldithio group. OX-26 was modified through its lysine amines withSATA (see Example 13) to introduce a protected sulfhydryl group forsubsequent reaction to form the disulfide bonds. The extent of proteinmodification was monitored and the reaction conditions were such thatthe number of groups attached to both the antibody and NGF was kept to aminimum (approximately 5 and 1, respectively). Conjugates were preparedby reacting derivatized NGF with the deprotected OX-26 at a 7.5:1 molarratio. The reactions were run under nitrogen in 0.1M sodium phosphate/3mM EDTA, pH 7.5 at room temperature for 2 hours. The free sulfhydrylgroup on the OX-26 exchanges with the 2-pyridyl-sulfide group on NGF,forming a disulfide bond between the two proteins and releasingpyridine-2-thione. In general, these reaction conditions generated a 1:1to 1:2 (OX-26:NGF) conjugate with a yield (in terms of NGF) of between20 and 30%.

Purification of these conjugates from unreacted starting materials wasinitiated by passage over a Protein-A Sepharose column to removeunreacted NGF. The material eluted from this column, which consisted ofconjugate and free antibody, was then passed over a NGF affinity column.Free OX-26 flowed through this column whereas OX-26-NGF conjugate wasretained.

The purity of the conjugate eluted from the NGF affinity column wasassayed using SDS-PAGE under reducing and nonreducing conditions.Conjugate formation was verified by ELISA and, on occasion, byimmunoblot or autoradiography as well. The activity of the NGF portionof the conjugate was verified by using the PC-12 neurite outgrowthassay. Typically, the conjugate activity was within one dilution of theNGF standard.

BRAIN TARGETING AND DELIVERY

The targeting of the antibody-NGF conjugate to the brain capillaries wasassessed using immunohistochemistry as described in Example 1. Anantimouse IgG antisera was used to localize the OX-26 portion of theconjugate while an anti-NGF antibody was used to localize the"passenger" protein. Both proteins were detected in the vasculature ofthe brain following iv administration of OX-26-NGF conjugate; NGF wasnot detected when injected as a free protein. That is, NGF was detectedin the vasculature of the brain only when it was administered in theform of a covalent conjugate with OX-26. In addition, the attachment ofNGF to OX-26 did not significantly alter the targeting of the antibodyto the brain capillaries.

The delivery function of the anti-TfR portion of the conjugate wasquantitated using the capillary depletion procedure as described inExample 9. For these studies, NGF was radiolabeled with ³ H prior toconjugation. The results indicated that approximately 0.5% of theinjected dose of NGF crossed the blood-brain barrier and accumulated inthe brain parenchyma when the NGF was conjugated to intact OX-26antibody. When NGF was conjugated to OX-26 Fab fragments, approximately0.15% of the injected dose of NGF crossed the blood-brain barrier andaccumulated in the brain parenchyma. When NGF was conjugated to OX-26F(ab')₂ fragments, between 0.1% and 0.2% of the injected dose of NGFreached the brain parenchyma after passing across the blood-brainbarrier.

BRAIN UPTAKE OF OX-26-NGF

Quantitative uptake into the brain of the OX-26-NGF conjugate over ashort-term time course was measured. These measurements demonstrated thepassage of the conjugate across the blood-brain barrier.

The measurements were performed by a capillary depletion proceduresimilar to that described in Example 9. In this procedure, a two-siteimmunoassay (EIA) for NGF was used. After dissection, the brain sampleswere frozen on dry ice, homogenized and centrifuged, and thesupernatants taken for EIA. Immunoplates were coated with monoclonalanti-mouse-β-NGF antibodies 27/21 (0.5 ug/ml, Boehringer-Mannheim).Control wells were coated with the same amount of control mouse IgG. Thedishes were incubated with the samples at 4° C. overnight. As standards,samples of purified mouse NGF were used. After extensive wash,27/21-β-galactosidase conjugate antibodies were applied overnight(Boehringer, 4 units of enzyme activity/ml). Enzyme activity wasmeasured in a microplate fluorometer (Dynatech Microfluor) and the NGFconcentration in brain samples determined by comparison with theregression line of the NGF standards. Statistical analysis was carriedout using analysis of variance in a general linear model (GLM) withrepeated measures or comparing small series of values with aMann-Whitney U-test.

Quantitative uptake into the brain was followed in the cerebellum, whichis a region of the brain with a low endogenous level of NGF, therebyfacilitating detection of the extra contribution from the injectedconjugate. For comparison, the uptake of injected NGF in thesubmandibular gland of the rat was followed as representing a peripheralorgan outside the BBB. As can be seen in FIG. 8, the levels of NGF weresignificantly increased in cerebellar tissue 4-8 hours followingintravenous injection of OX-26-NGF, but not OX-26, saline or NGF alone.These results indicate that it takes some time for the OX-26-NGFconjugate to clear from the vascular wall, and to be taken up by thebrain tissue (compare the levels at 1 hour postinjection). Also, it isclearly shown in this figure that equimolar injection of NGF alone didnot result in any significant uptake of NGF into the cerebellum.

FIMBRIA FORNIX LESION MODEL

Cholinergic neurons, which are localized in the nucleus basalis ofMeynert and the medial septal nucleus, innervate the neocortex,hippocampus and amygdala in response to the NGF produced by thesepostsynaptic target areas. It has been shown in rats that if the supplyof NGF is interrupted by transection of the fimbria fornix, whichresults in a lesion in the septo-hippocampal pathway, the cholinergicneurons in the basal forebrain will degenerate. If exogenous NGF isadministered by intracerebroventricular (icy) infusion to the lesionedanimals, the atrophy by cholinergic neurons can be prevented. (Williamset al., "Continuous Infusion of Nerve Growth Factor Prevents BasalForebrain Neuronal Death after Fimbria Fornix Transaction", PNAS 83,9231-9235 (1986)).

This model system was used to examine the efficacy of NGF delivered ivusing an anti-TfR antibody. The treatment groups in this experimentconsisted of OX-26-NGF iv, NGF iv, OX-26 iv, NGF icv and carrier buffericy. The iv dosing regimen consisted of 30 μg NGF, either as conjugateor free protein, or an amount of OX-26 equivalent to that in theconjugate, given daily for two weeks. Based on the percent of theinjected dose of NGF that reaches the brain parenchyma as determinedfrom the capillary depletion experiments, this dose of conjugate shouldresult in the delivery of approximately 2.0 μg of NGF. Taking intoaccount the K_(D) of the NGF receptor, this amount of NGF should be morethan sufficient to stimulate the cholinergic neurons of the basalforebrain if the NGF is transported intact across the blood-brainbarrier. The icv animals were treated via cannulas attached to Alzetosmotic minipumps.

At the completion of the dosing period, the animals were perfused andthe brains removed for histochemical analysis. Acetylcholinesterase(ACHE) histochemistry was used to verify the completeness of the lesion.Immunohistochemical staining using an antibody to the low affinity NGFreceptor (LNGFR or P75) was used to visualize the cholinergic neurons inthe basal forebrain. Neurons on the ipsilateral side of the lesionatrophy due to a lack of NGF. The results were scored by counting thenumber of neurons remaining on the lesioned (ipsilateral) side of thebrain relative to the number on the unlesioned (contralateral) side.Typically between 50-60% of the neurons on the lesioned side of thebrain degenerate without an exogenous source of NGF.

The results of this experiment are shown in Table 1.

                  TABLE I                                                         ______________________________________                                        Results of Fimbria Fornix Experiment #1                                                          Result (% stained neurons,                                 Treatment          ipsilateral/contralateral)                                 ______________________________________                                        NGF iv              41.5 ± 8.3                                             carrier buffer icv   50.0 ± 10.5                                           AK-26-NGF conjugate iv (#1)                                                                      60.8 ± 19                                               AK-26-NGF conjugate iv (#2)                                                                      96.5 ± 32                                               AK-26-NGF conjugate iv (#3)                                                                      85.9 ± 14                                               ______________________________________                                    

In the two negative control animals that had complete lesions (1 NGF ivand 1 carrier buffer icv), approximately 40-50% of the neurons on theipsilateral side of the lesion survived relative to those on thecontralateral side of the lesion. In contrast, the percent of neuronssurviving in two of the conjugate-treated animals was ˜80-90%; this isas good as, if not better than, what has been reported for NGF treatmenticv. A third conjugate treated animal showed ˜60% neuron survival, whichwas less than that for the other two animals but still better than thenegative controls.

EXAMPLE 16

Delivery of Nerve Growth Factor to Intraocular Implants viaAnti-Transferrin Receptor Antibodies

Another method for analyzing the ability of the anti-TfR antibodies todeliver active NGF across the blood-brain barrier is to examine theeffect of OX-26-NGF conjugates on fetal medial septal nucleus tissuethat has been implanted into the anterior chamber of the eye. Thissystem provides a means for studying the effects of NGF on thedeveloping cholinergic neurons in the medial septal nucleus that hasbeen isolated from other CNS influences. An advantage of this system isthat cholinergic neurons in fetal tissue are much more sensitive to NGFthan are those in adult tissue.

To demonstrate the usefulness of this system for the study of NGFdelivery via the anti-rat TfR antibody, preliminary immunohistochemistryexperiments were performed to examine the localization of the antibodyin the tissue grafts. The results of these studies indicated that OX-26targets to the vasculature of the implants in a manner indistinguishablefrom that seen in the host brain.

In this experiment, 2 mm³ pieces of medial septal nucleus tissue weredissected from rat fetuses at gestational day E18 and bilaterallygrafted to the anterior chamber of the eye of adult rats (2 groups of 6animals each). NGF treatment was not begun until two weeks aftergrafting to allow vascularization of the implants and formation of avascular equivalent of the blood-brain barrier. The dose of conjugatethat was administered was calculated based on the size of the targettissue the percent of the injected dose of the conjugate NGF whichcrosses the blood-brain barrier and the K_(d) of the NGF receptor forNGF. These calculations resulted in a dose/injection of ˜12 μg NGF. Inkeeping with previous experiments which examined the effects ofintraocularly administered NGF on implants in the anterior chamber ofthe eye, the initial dosage regimen involved iv bolus injections ofconjugate or control every two weeks over a period of 8 weeks. Duringthis time period, the effect of the conjugate-derived NGF on the growthof the tissue implants was monitored by weekly observations through thecornea of the living host using a stereo-microscope equipped with aneyepiece micrometer.

The results of the implant growth studies are shown in FIG. 9. Duringthe 2 weeks prior to the first treatment, the size of the implantsdecreased steadily. Coincident with the first treatment, the two groupsdiverged with the OX-26-NGF conjugate-treated implants stabilizing insize and the OX-26-treated implants continuing to decrease in size. Inaddition, the conjugate-treated grafts appeared to increase in sizeafter the 6 week injection. These results suggest that the NGF deliveredby the conjugate sustains the cholinergic neurons in the graft, thusleading to the increased graft size relative to the control.

At the completion of the dosing period, the animals were sacrificed sadthe tissue fixed, sectioned and processed for ChAT-immunostaining toidentify cholinergic cells; it had been shown previously thatintraocular administration of NGF can increase the number ofChAT-positive cells in basal forebrain tissue grafts by approximately80% over that observed in untreated control grafts . The results fromthe grafts taken from OX-26 treated animals indicated little or nostaining for ChAT in this tissue, suggesting that few, if any,cholinergic neurons had survived. In contrast, grafts taken fromOX-26-NGF treated animals showed very intense ChAT staining with manyChAT positive cells clearly visible. These results support tire resultsof the implant growth measurements anal indicate that the conjugate isable to deliver active NGF to the tissue grafts, which possess avascular equivalent to an intact blood-brain barrier.

Despite the marked differences in overall graft sizes, histologicalexamination of the septal transplants showed no obvious differences inthe density of neural or glial structures within the two groups ofgrafts. Specifically, the density of cells and the vascularization ofgrafts in the two groups appeared to be the same. To specificallyinvestigate the survival of septal cholinergic neurons in thetransplants, the CHAT-immunohistochemistry was used to reliablyvisualize cholinergic structures in nervous tissue. A much larger numberof cholinergic neurons, approximately a three and a half-fold increase,was found in the group of transplants injected with OX-26-NGF ascompared to the control group. Because the transplants in the NGFconjugate groups were significantly larger, the total numbers of bothcholinergic and non-cholinergic neurons surviving were larger than inthe control groups, a finding in keeping with previous studies that madeuse of intraocular injections of NGF. These results further support theconcept that NGF passed through the blood-brain barrier into the graftsand enhanced the survival of cholinergic neurons in the groups injectedwith the NGF conjugate. These results also indicate that NGF passedthrough the blood-brain barrier into the grafts and enhanced thesurvival of non-cholinergic neurons, perhaps by indirect or accessaryprocesses, in the groups injected with the NGF conjugate.

EXAMPLE 17

Formation of Anti-Transferrin Receptor Antibody Conjugates withSuperoxide Dismutase Which Retain Enzyme Activity

Superoxide dismutase (SOD), a 32,000 dalton protein, scavengessuperoxide free radicals by converting them to hydrogen peroxide andmolecular oxygen. It has been shown that superoxide radicals, which areproduced during ischemia and reperfusion, alter endothelial cell andblood-brain barrier permeability and elicit brain edema and cellularinjury through lipid peroxidation. The primary difficulties associatedwith using SOD as a therapeutic for stroke have heretofore been itsshort serum half-life and poor localization at the site of damage.

CONJUGATE SYNTHESIS AND PURIFICATION

Conjugation of superoxide dismutase (SOD) to OX-26 was performedfollowing the strategy previously described for CD4 (see Example 13).SOD was modified with SATA to introduce a protected thiol group andOX-26 was modified through the carbohydrate group with PDP-hydrazide toyield a disulfied-linked conjugate. The yield of conjugate with thisapproach was approximately 10% (in terms of SOD).

Purification of the conjugate from unreacted SOD was achieved by passingthe material over a gel filtration column. Chromatography on a Mono Scolumn (a cation exchanger) separated the free SOD and some of the freeantibody from the conjugates. This was accomplished by recovering onlythe leading edge of the antibody elution from the column. Approximatelya 50:50 mixture of conjugate and free antibody was obtained. OX-26-SODconjugate synthesis was verified by immunoblot and by ELISA.

The activity of SOD after conjugation was measured using a dye reductionassay. When riboflavin is photochemically reduced and then re-oxidizedthrough exposure to air, superoxide radicals are produced. Theseradicals then reduce nitroblue tetrazolium dye to a blue color. Theassay measures the ability of SOD to inhibit the reduction of the dye byremoving the superoxide radicals. It was found that the recoveredOX-26-SOD conjugates retained greater than 60% of the SOD activity.

EXAMPLE 18

Formation of Anti-Transferrin Receptor Antibody Conjugate withAnti-Amyloid Antibody

Anti-amyloid antibodies or antibody fragments immunoreactively bind withamyloid plaques that are characteristic of Alzheimer's Disease. Forclinical diagnostic detection purposes, a detectable group such asgadolinium or technitium is often attached to the anti-amyloid antibody.This allows imaging of the brain to be performed. Heretofore, it hasbeen difficult to easily deliver a diagnostically useful amount of theanti-amyloid antibodies past the blood-brain barrier.

CONJUGATE SYNTHESIS AND PURIFICATION

The two antibodies (OX-26 and 10H3) were joined together using SATA andSPDP to form a disulfide linkage following procedures describedpreviously (Example 13). The conjugate (approximately 1:1) was purifiedfrom free antibody on a gel filtration column. Some of this conjugatewas tested both by slot-blots and ELISA for binding to the amyloidpeptide used to produce the antibody. The antigen binding activity ofthe conjugate was as good as, if not better than, the unconjugatedantibody; this indicated that the 10H3 antibody was not affected by theconjugation process. In addition, the conjugate was injected into a ratand immunohistochemistry was used to examine targeting to the brainvasculature via the anti-transferrin receptor antibody. The conjugatewas localized to the brain vasculature in a manner similar to that forOX-26 alone, indicating that the attachment of this anti-amyloidantibody to OX-26 did not affect its ability to target to braincapillary endothelial cells in vivo

Equivalents

Those skilled in the art will know, or be able to ascertain using nomore than routine experimentation, many equivalents to the specificembodiments expressly described herein. These are intended to be withinthe scope of the invention as described by the claims herein.

I claim:
 1. A method for delivering a therapeutically effective amountof a nerve growth factor or a neurotrophic factor across the blood-brainbarrier of a mammal comprising administering a conjugate of an antibody,or biologically functional binding fragment thereof, and either a nervegrowth factor or a neurotrophic factor to the mammal under conditionswhereby said conjugate binds to transferrin receptors on brainendothelial cells and the nerve growth factor or the neurotrophic factoris transported across the blood-brain barrier of the mammal in apharmaceutically active form and in a therapeutically effective amount.2. A method of claim 1 wherein the neurotrophic factor is selected fromthe group consisting of ciliary neurotrophic factor, brain-derivedneurotrophic factor, neurotrophin 3, neurotrophin 4 and neurotrophin 5.3. A method of claim 1 wherein said antibody comprises a monoclonalantibody.
 4. A conjugate of an antibody, or biologically functionalbinding fragment thereof, and either a nerve growth factor or aneurotrophic factor wherein said conjugate is bindable to transferrinreceptors on brain endothelial cells.
 5. A conjugate of claim 4 whereinthe neurotrophic factor is selected from the group consisting of ciliaryneurotrophic factor, brain-derived neurotrophic factor, neurotrophin 3,neurotrophin 4 and neurotrophin
 5. 6. A conjugate of claim 4 whereinsaid antibody comprises a monoclonal antibody.
 7. A pharmaceuticalcomposition comprising: (a) a conjugate of an antibody, or biologicallyfunctional binding fragment thereof, and either a nerve growth factor ora neurotrophic factor, and (b) a pharmaceutically acceptable carrier,wherein said conjugate is bindable to transferrin receptors on brainendothelial cells.
 8. A composition of claim 7 wherein the neurotrophicfactor is selected from the group consisting of ciliary neurotrophicfactor, brain-derived neurotrophic factor, neurotrophin 3, neurotrophin4 and neurotrophin
 5. 9. A composition of claim 1 wherein said antibodyportion comprises a monoclonal antibody.